Goals and Strategy: This project seeks to develop individualized (patient-specific) therapies that will use antibodies and/or immune cells ("natural killer cells," macrophages, cytotoxic T cells) to attack cancer-causing leukemia "stem" cells (LSC's) without harming normal (blood) hematopoietic "stem" cells (HSC's). Lab activities are designed to (1) isolate and compare LSC's and normal HSC's from the same patient, (2) identify immunologic "targets" located only on the cancer-causing LSC's, and (3) explore immune-therapy options suggested by cell-structure differences. Imaging is vital and will include light microscopy, confocal (3-D), electron (transmission and scanning), and two-photon (3-D). When enhanced by staining techniques, cell micrographs can identify intracellular and surface structures as well as cell type and maturity level. Regarding growing, transforming, and analyzing cells, this proposal is essentially a logical extension of recent (and profound) stem-cell-related achievements. New insights and technical advances provide all the tools needed to develop immune-therapies: a useful conceptual framework, ways to transform cells, and high-tech lab and imaging resources. These advances inspired us to grow "blood cell-spheres" (BCS) using leukemia cells taken from routine patient blood specimens. These new leukemia BCS are exciting because other "cancer cell-spheres" are known to contain, concentrate, or "create" those critical cancer-causing "stem" cells that are rare and elusive inside a patient's body. Now, normal BCS will be grown (exciting since normal-cell spheres like "blastemas" also contain "stem" cells - these regenerate amputated limbs in animals). BCS will be imaged and analyzed to determine if (1) LSC's are inside leukemia BCS and (2) normal HSC's are inside normal BCS. If found - and stem cells have been found in similar cell-spheres - LSC's and normal HSC's will be compared using staining/imaging techniques that can show internal and surface details of spheres and the individual cells inside them. If the desired "stem" cells are not present, "newer generations" of BCS will be grown after adding more factors and cells that promote "stem" cell creation. When both normal and cancer "stem" cells can be isolated, they will be carefully compared to identify potential "targets" found only on LSC's. Finally, antibodies and/or immune cells that can attack these unique targets will be developed. Health-Impact: If LSC's and normal HSC's can be isolated from BCS grown from a leukemia patient's blood specimen, patient-specific immunotherapies can be developed. Individualized therapies would eliminate treatment failures caused by genetic variations and would represent one of the most ideal and unassuming strategies possible. Also, any lab procedure that enables scientists to derive "stem" or "progenitor" cells from more mature cells may help scientists regenerate human tissues and organs. PUBLIC HEALTH RELEVANCE: This research seeks to develop blood-cancer therapies that use antibodies and/or special immune cells (like "natural killer cells," macrophages, cytotoxic T cells) to attack a patient's (mutant) leukemia-causing stem-cells without harming normal cells. From a routine blood specimen, normal and leukemia "blood cell-spheres" will be grown to concentrate or "create" both normal and cancer stem-cells. These critical cells will be compared to find structures only present on the leukemia-causing cells that can be targeted by immunotherapies.